HER-2/neu Expression in Resectable Gastric Cancer and its Relationship with Histopathologic Subtype, Grade, and Stage

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1 Iranian Journal of Basic Medical Sciences Vol. 10, No. 2, Summer 2007, Received: 18 Apr 2007, Accepted: 14 Aug 2007 HER-2/neu Expression in Resectable Gastric Cancer and its Relationship with Histopathologic Subtype, Grade, and Stage * 1 H.R. Raziee, 1 A. Taghizadeh Kermani, 2 K. Ghaffarzadegan, 3 M. Taghi Shakeri, 1 M.R. Ghavamnasiri Abstract Objective HER-2/neu is overexpressed in diverse human cancers. Studies suggest a role of this protein in tumor progression by specifically promoting the invasive capacity of tumor cells. Our aim was to evaluate HER- 2/neu overexpression in resectable gastric cancer in 100 North-Eastern Iranian patients and to assess the relationship between its expression and clinicopathologic tumor parameters. Materials and Methods Indirect immunostaining was employed to evaluate the expression of this receptor in formalin-fixed paraffin-embedded tissue samples. Results HER-2/neu overexpression was present in 26 (26%) of 100 gastric carcinomas. This was significantly more common in the intestinal type of gastric cancer (33%) compared to diffuse (5%) or the mixed type (0%). HER-2/neu overexpression was also more common in well-differentiated gastric cancers versus other grades (41% vs 7%). However, it was not associated with gender, age at diagnosis or stage. Conclusion HER-2/neu overexpression is common in gastric carcinoma and more prevalent in intestinal and welldifferentiated subtypes. There is no correlation between HER-2/neu expression and tumor stage. The relatively high percentage of HER-2/neu positive tumors may provide a useful target for immunotherapy of these cancers. Keywords: HER-2/neu, Gastric cancer, Grade, Immunohistochemistry, Lauren's classification. 1- Radiation Oncology Department, Cancer Research Center, Omid Hospital, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran *Corresponding author: Tel: ; Fax: ; razieehr@mums.ac.ir 2- Pathology Department, Cancer Research Center, Omid Hospital, MUMS, Mashhad, Iran 3- Biostatistics Department, MUMS, Mashhad, Iran IJBMS, Vol. 10, No. 2, Summer

2 HER-2/neu Expression in Resectable Gastric Cancer Introduction HER-2/neu oncogene, also known as c-erbb- 2, encodes a transmembrane tyrosine kinase receptor, homologous to epidermal growth factor receptor (1). HER molecules belong to a family of glycoproteins that consist of an extracellular domain for binding ligands, a short lipophilic transmembrane domain, and an intracellular domain carrying tyrosine kinase activity. HER-2/neu gene, located on chromosome 17q21, is related to the oncogene v-erbb of the avian erythroblasosis virus. The protein encoded by this gene p185 is suggested to be a growth factor receptor involved in the growth and progression of malignant cells. However, the ligand of p185 is not yet identified (2). HER-2/neu gene is found to be amplifiend in 10% to 30% of human breast, ovarian, gastric and other diverse cancers including lung adenocarcinoma, uterine cervix carcinoma and squamous cell carcinomas of the head and neck (3-6). Depending on the methodological aspects of the assays, scoring criteria and case selection, the prevalence of HER-2/neu expression shows wide variation in different studies on various tumors. The prognostic value of c-erbb-2 has been primarily shown in breast cancer where patients with overexpression of this gene have a significantly lower relapse free and overall survival rate than patients without overexpression (7-12). Evidence also exists in gastric cancer showing that overexpression of this protein is a new, independent prognostic factor for overall survival (13). However, some studies have failed to find an association with prognosis (14-17). On the other hand, with the availability of the monoclonal antibody trastuzumab, HER-2/neu can be the target of therapy in this disease, adding to the importance of research on HER-2. (18). Immunohistochemistry and fluorescence in situ hybridization (FISH) are the techniques routinely recommended for determining HER-2/neu status, in terms of protein overexpression and genomic amplification, respectively (18). The aims of this study are: 1) to assess HER-2/neu content in our gastric carcinoma patients and 2) to assess the correlation between this receptor tumor content and clinicopathologic characteristics. Patients and Methods One hundred gastric adenocarcinoma pathologic specimens from patients who had undergone curative surgery between 2000 and 2006 were identified from the specimen archive, Pathology Department, Omid University Hospital, Mashhad, Iran. All patients had been residing in North Eastern Provinces of Iran at the time of surgery. TNM staging had been performed on all patients according to AJCC (American Joint Committee on Cancer) staging before treatment. No patient had received chemotherapy or radiation therapy before curative resection. All patients had undergone staging procedures by supra- and infra- diaphragmatic imaging studies, i.e. chest X-ray and abdominal ultrasonography or CT scanning. Immunohistochemistry A section from specimen blocks was stained with H&E for histological subtype evaluation and the grade was determined. Representative blocks were chosen for immunostaining. Four-micrometer-thick sections were dewaxed and processed for immunohistochemistry. IHC was performed with the HercepTest kit (k5204, Dakocytomation, Denmark) according to manufacture s instructions. Diaminobenzidine was used as the chromogen, and the slides were counterstained with hematoxylin. We used known positive breast cancer cases as 140 IJBMS, Vol. 10, No. 2, Summer 2007

3 H.R. Raziee positive control. For negative control we omitted the primary antibody. All sections were evaluated by our pathologist without knowledge of the clinical characteristics of the patients. Tumors with more than 10% of cancer cells showing membranous staining for HER-2 were classified as positive. Cytoplasmic staining was not accounted. The results were reported according to the Dako manual for interpretation of HER-2 staining, a semiquantitative system based on intensity of cell-membrane immunostaining and the percentage of positive cells in the suitable carcinoma fields (score range from 0 to 3+). In this study, as in breast cancer, scores 2 and 3 of IHC intensity were assumed to be positive for overexpression in any percentage of cells (Figures 1-3). Figure 1. HER-2/neu scores 3+ overexpression. (40x magnification) Figure 3. HER-2/neu score1+ overexpression. (40x magnification) The relation between the HER-2/neu staining and clinicopathologic variables was analyzed. SPSS 11.5 was used for statistical analysis. p values were calculated by using Chi-square, Fisher exact test, t test and ANOVA. Figure 2. HER-2/neu scores 2+ overexpression. (40x magnification) Results The mean age was years (from 26 to 82) and the male/female ratio was 2.8 (74 men, 26 women). 3% of cases were stage I, 13% stage II, 64% stage III, 20% stage IV. According to Lauren s classification, 74 tumors (74%) were intestinal, 21 (21%) were diffuse and five (5%) were mixed-type carcinomas. 54 tumors were well differentiated, (Grade 1), 17 were moderately-differentiated (Grade 2) and 29 were poorly-differentiated (Grade 3). From 100 tumors, 36 were located at the cardia, one at the fundus, 33 at the corpus and 30 in the antrum. The positive staining with IHC method ranged from 0 to 90 percent of tumoral cells. In 26 out of 100 samples (26%) HER-2/neu protein overexpression was observed. Positive reactivity with anti c- IJBMS, Vol. 10, No. 2, Summer

4 HER-2/neu Expression in Resectable Gastric Cancer erbb-2 antibody was significantly more frequent in low grade (differentiated) tumors rather than high grade (undifferentiated) tumors (p=0.001) (Table 1). Table 1. Relationship between HER-2/neu expression and clinicopathologic features in patients with gastric cancer Cilnicopathologic data n HER-2/neu pvalue pos % Sex Male female Age >65 <65 T T1 T2 T3 T4 N N0 N1 N2 N % 6 23% 15 28% 11 23% 1 25% 21 26% 4 23% 5 31% 16 31% 4 13% 1 33% Location of tumor Cardia Fundus Body Antrum Histology Intestinal Diffuse Mixed Differentation Well-diff Mod-diff Poorly-diff % 9 27% 7 23% 25 33% 1 5% 22 41% 2 11% 2 7% p=0.001 p=0.001 Stage of cancer M M0 M % 16 25% 4 20% 26 27% - n: number, pos: positive, T: Tumot, N: Node, M: Metastasis Statistical Analysis A significantly higher expression of c-erbb-2 was observed in tumors with intestinal histology compared to diffuse or mixed types (33% vs 5% vs 0%, p=0.001). With the exception of tumor grade and tumor histology, no other significant correlations were observed between overexpression of this protein and studied variables (Table1). The mean age in patients with poorly 142 IJBMS, Vol. 10, No. 2, Summer 2007

5 H.R. Raziee differentiated tumors was lower than other patients (p=0.019). Besides, the mean age in patients with HER-2/neu positive tumors was lower than patients with negative HER-2/neu (p=0.001). Stage IV disease was significantly more common in patients younger than 65 years and all stage I cases were observed among patients older than 65. The number of patients with diffuse histology was two times higher in patients under 65 yrs old. Discussion Tumors of the upper gastrointestinal tract have been reported to show a wide range of over expression of HER-2/neu protein (19). In the present study we investigated the expression of this receptor protein in 100 Iranian gastric cancer patients, retrospectively. Our observed prevalence of HER-2/neu protein over expression by immunohistochemistry (26%) fits the range previously reported ( %) (20). However, at the upper range there are some limitations inherent in IHC technique, such as those caused by variability in fixation, and the problems in standardization and scoring of the staining. In a FISH-based study recently reported, HER-2/neu was found to be amplified in about (18%) of the cases (21). Our percentage can be modified as well if we employ FISH method the state-of-theart alternative to IHC on the same samples. This is designed as the future direction of this project. As in breast carcinoma, where HER-2/neu gene amplification is common in invasive ductal carcinoma and an uncommon feature in lobular carcinoma, the intestinal type of gastric cancer shows higher prevalence of HER-2/neu amplification in contrast to diffuse type (21). Our results also confirm that HER-2/neu protein over expression is strongly associated with the intestinal histological subtype as defined according to Lauren s classification (33% in intestinal versus 5% in diffuse subtype, P= 0.001). In other three studies (22-24), immunohistochemical HER- 2/neu overexpression has been as well more common in the intestinal rather than diffuse type of gastric adenocarcinoma. However, so far it has been difficult to evaluate the clinical importance of this finding. In this study, well differentiated tumors were more likely to overexpress the HER- 2/neu protein. This is in agreement with the results reported by other investigators (25, 26). HER-2/neu gene amplification has been associated with the degree of differentiation of adenocarcinomas, in that well differentiated adenocarcinomas have shown a very high incidence (up to 100%) of HER- 2/neu amplification (27, 28). However, not all studies agree, and a positive relation with poor tumor differentiation has been reported as well (29). In this work, regarding previous published studies (19, 30-32), we considered HER- 2/neu over expression as a prognostic factor, depicting a poor prognosis. On the other hand, surgical stage is by itself another proven prognostic factor. Our aim was to establish a correlation between HER-2/neu expression status and stage. The absence of this relation in our results might be due to the fact that HER-2/neu positive tumors have a more aggressive pathologic behavior, which is reflected in more common micrometastatic disease at presentation and more distant failures later after treatment. Additionally, it should be noted that there are inconsistencies in the literature regarding the clinical significance of HER-2 over expression which may be due to problems of selecting the best methodology, standardizing the methods, and determining the correct cut off points. The present drug sensitivity assays suggest that the presence of HER-2/neu amplification may turn out to be a more important feature in therapeutic prediction than in prognostication in gastric adenocarcinomas (21). Analysis of a HER-2/neu amplified gastric carcinoma cell line N87 indicated that IJBMS, Vol. 10, No. 2, Summer

6 HER-2/neu Expression in Resectable Gastric Cancer it was as sensitive to trastuzumab as the HER-2/neu amplified breast carcinoma cell line SKBR-3, which is a widely used reference in trastuzumab sensitivity studies (33, 34).This will open the doors to new treatment approaches in gastric cancer in future. Future Direction Regarding the high prevalence of gastric cancer in this country and its poor outcome, the importance of research on novel prognostic and therapeutic measures can not be overlooked. With increasing the sample size in future studies and designing prospective studies with close observation of survival, we will be trying to determine the prognostic role of HER-2/neu over expression. Besides, FISH or CISH methods will be utilized to detect the gene amplification. Acknowledgements This work was financially supported by a grant from Mashhad University of Medical Sciences, MUMS. We would also like to thank Ms As adi for her laboratory assistance. References 1. Press MF, Cordon-Cardo C, Slamon DJ. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissue. Oncogene 1990; 5: Yamamoto T, Ikawa S, Akiyama T. Similarity of protein encoded by the human c-erbb-2 gene to epidermal growth factor receptor. Nature 1986; 319: Bongiorno PF, Whyte RI, Lesser EJ. Alterations of K-ras, p53, and erbb-2/neu in human lung adenocarcinomas. J Thorac Cardiovasc Surg 1994; 107: Mitra AB, Murty VVS, Pratap M. ERBB2 (HER-2/neu) oncogene is frequently amplified in squamous cell carcinoma of the uterine cervix. Cancer Res 1994; 54: Slamon DJ, Clark GM, Wong SG. Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/ neu oncogene. Sci 1987; 235: Beckhardt RN, Kiyokawa N, Liu TJ. Neu oncogene in head and neck squamous cell carcinoma. Proc Am Assoc Cancer Res 1994; 35: Hynes N, Stern D. The biology of erbb-2/neu/her-2 and its role in cancer. Biochim Biophys Acta 1994; 1198: Slamon DJ, Clark GM, Wong SG. Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/ neu oncogene. Sci 1987; 235: Guerin M, Gabillot M, Mathieu MC. Structure and expression of c-erbb-2 and EGF receptor genes in inflammatory and non-inflammatory breast cancer: Prognostic significance. Int J Cancer 1989; 43: Van de Vijiver MJ, Peterse JL, Mooi WJ. Neu protein overexpression in breast cancer: Association with comedo type-ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N Engl J Med 1988; 319: Berger MS, Locher GW, Sauer S. Correlation of c-erbb-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading, Cancer Res 1988; 48: Paik S, Bryant J, Tan-Chiu E, Yothers G, Park C, Wickerham DL, et al. HER2 and choice of adjuvant chemotherapy for invasive breast cancer: National Surgical Adjuvant Breast and Bowel Project Protocol B-15. J Natl Cancer Inst 2000; 92: Allgayer H, Babic R, Gruetzner UG, Tarabichi A, Schildberg FW, Heiss MA. C-erbB-2 is of independent prognostic relevance in gastric cancer and is associated with the expression of tumor-associated protease system. J Clin Oncol 2000; 18: Hilton D, West K. c-erbb-2 oncogene product expression and prognosis in gastric carcinoma. J Clin Pathol 1992; 45: Kolodziejczyk P, Yao T, Oya M. Long term follow-up study of patients with gastric adenomas with malignant transformation. Cancer 1994; 74: IJBMS, Vol. 10, No. 2, Summer 2007

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